Project description:We report the application of next-generation sequencing (NGS) technology for high-throughput profiling of RNA expression in human breast cancer cells. The goal of this study was to compare NGS-derived transcriptome profiling (RNA-seq) from cells expressing either wildtype or knockout CD44 to identify key downstream signaling events of HA-CD44 interactions. Methods: This experiment was repeated for a total of three times and replicate RNA samples were extracted and submitted for Illumina Sequencing. Twelve dual-indexed Illumina TruSeq stranded mRNA libraries were pooled for sequencing on 1 lane of a HiSeq 2500 using v4 chemistry to generate 2x50 bp paired-end reads. After applying the Benjamini-Hochberg multiple hypothesis adjustment to raw p-values, differentially expressed genes were identified requiring that they had a log2 fold change > 0 and an adjusted p-value ≤ 0.01. Results: A total of 1,530 and 320 genes were impacted by CD44 KO in the Hs578T cells and MDA-MB-231 cells, respectively. When comparing each of these data sets, 40 genes were differentially expressed in both Hs578T and MDA-MB-231 cells upon deletion of CD44. Conclusions: Gene ontology (GO) analysis revealed that CD44 KO in breast cancer cells altered expression of genes involved in both cell adhesion and cytokine activity. This study provides a framework for the application of NGS to compare differential gene expression between genetically edited mammalian cell populations.
Project description:CD44, an adhesion molecule that binds to extracellular matrix, primarily to hyaluronan (HA), has been implicated in cancer cell migration, invasion, and metastasis. CD44 has also recently been recognized as a marker for stem cells of several types of cancer. However, the roles of CD44 in the development of bone metastasis still remain unclear. To explore this issue, we established the MDA-MB-231 human breast cancer cells stably expressing short hairpin RNA against CD44. The CD44-knockdown MDA-MB-231 cells (MDA-MB-231 shCD44-2 and shCD44-3) were analyzed. As control, MDA-MB-231 cells stably expressing shRNA against firefly luciferase (shLuc) were used. Total of three samples. No replicates.
Project description:CD44, an adhesion molecule that binds to extracellular matrix, primarily to hyaluronan (HA), has been implicated in cancer cell migration, invasion, and metastasis. CD44 has also recently been recognized as a marker for stem cells of several types of cancer. However, the roles of CD44 in the development of bone metastasis still remain unclear. To explore this issue, we established the MDA-MB-231 human breast cancer cells stably expressing short hairpin RNA against CD44.
Project description:CD44 expression has been shown to be enhanced in the liver and white adipose tissue (WAT) during obesity, suggesting a possible regulatory role for CD44 in metabolic syndrome. To study this hypothesis, we compared the gene expression profiles in liver and in WAT between WT and CD44 knockout (CD44KO) mice fed a high-fat diet (HFD) for 21 weeks. This analysis demonstrated that several genes associated with triglyceride synthesis and accumulation, including Mogat2, Cidea, Cidea, Apoa4, and Elovl7, were decreased in the livers of CD44KO mice compared to WT mice. Many genes encoding pro-inflammatory chemokines and chemokine receptors also were decreased in the livers of CD44KO mice. Analysis with WAT showed that genes associated with triglyceride accumulation, including Fasn, Elovl6 and Mogat2, were increased in WAT of CD44KO(HFD) mice compared to WT(HFD) mice. Moreover, many genes associated with inflammation, including cytokines (Cxcl14, Cxcl12, Il33, and Il2), cytokine receptors (Ccr1, Il6ra, Il10rb), trypases (Tpsb2, Tpsab1, Tpsg1), and cellular matrix proteins (Integrin ?4 (Itga4), ItgaM, Itgb2), were decreased in WAT of CD44(HFD) compared to WT(HFD) mice. This study indicates that CD44 plays a critical role in regulating several aspects of metabolic syndrome. Liver and white adipose tissue (WAT) total RNAs were purified from 5 WT and 5 CD44 knockout mice fed with a high-fat diet for 21 weeks. Then, samples were applied on Agilent mouse genome chips.
Project description:Osteosarcoma is the most common type of pediatric bone tumor. Despite great advances in chem-otherapy during the past decades the survival rates of osteosarcoma patients remains usatisfacto-ry. Drug resistance is one of the main reasons leading to treatment failure and poor progno-sis. Previous reports correlated expression of cluster of differentiation 44 (CD44) with drug re-sistance and poor survival of osteosarcoma patients, however the underlying mechanisms are poorly defined. Here, we investigated the role of CD44 in the regulation of drug chemoresistance using osteosarcoma cells isolated from mice carrying a mutation of the tumor suppressor neurofi-bromatosis type 2 (Nf2) gene. CD44 expression was knocked-down in the cells using CRISPR/Cas9 approach. Subsequently, CD44 isoforms and mutants were re-introduced to investigate CD44-dependent processes. Sensitivity to doxorubicin was analyzed in the osteosarcoma cells with modified CD44 expression by immunoblot, colony formation- and WST-1 assay. To dissect the molecular alterations induced by deletion of Cd44, RNA sequencing was performed on Cd44-positive and Cd44-negative primary osteosarcoma tissues isolated from Nf2-mutant mice. Subsequently, expression of candidate genes was evaluated by quantitative reverse transcription PCR (qRT-PCR). Our results indicate that CD44 increases the resistance of osteosarcoma cells to doxorubicin by up-regulating the levels of multidrug resistance (MDR) 1 protein expression, and suggest the role of proteolytically released CD44 intracellular domain, and hyaluronan interac-tions in this process. Moreover, high throughput sequencing analysis identified differential regula-tion of several apoptosis-related genes in Cd44-positive and -negative primary osteosarcomas, in-cluding p53 apoptosis effector related to PMP-22 (Perp). Deletion of CD44 in osteosarcoma cells led to doxorubicin-dependent p53 activation and a profound increase in Perp mRNA expression. Overall, our results suggest that CD44 might be an important regulator of drug resistance and suggest that targeting CD44 can sensitize osteosarcoma to standard chemotherapy.
Project description:Investigation of the role of hyaluronan in cardiomyocytes . The aims of the study were to determine if cardiomyocytes are capable of detecting hyaluronan and if then intrecellular signaling causes changes in gene expression. The study showed that cardiomyocytes can bind hyaluronan with following change in gene expression.
Project description:CD44 expression has been shown to be enhanced in the liver and white adipose tissue (WAT) during obesity, suggesting a possible regulatory role for CD44 in metabolic syndrome. To study this hypothesis, we compared the gene expression profiles in liver and in WAT between WT and CD44 knockout (CD44KO) mice fed a high-fat diet (HFD) for 21 weeks. This analysis demonstrated that several genes associated with triglyceride synthesis and accumulation, including Mogat2, Cidea, Cidea, Apoa4, and Elovl7, were decreased in the livers of CD44KO mice compared to WT mice. Many genes encoding pro-inflammatory chemokines and chemokine receptors also were decreased in the livers of CD44KO mice. Analysis with WAT showed that genes associated with triglyceride accumulation, including Fasn, Elovl6 and Mogat2, were increased in WAT of CD44KO(HFD) mice compared to WT(HFD) mice. Moreover, many genes associated with inflammation, including cytokines (Cxcl14, Cxcl12, Il33, and Il2), cytokine receptors (Ccr1, Il6ra, Il10rb), trypases (Tpsb2, Tpsab1, Tpsg1), and cellular matrix proteins (Integrin α4 (Itga4), ItgaM, Itgb2), were decreased in WAT of CD44(HFD) compared to WT(HFD) mice. This study indicates that CD44 plays a critical role in regulating several aspects of metabolic syndrome.
Project description:Objectives/Design: Intervertebral Disc (IVD) degeneration has been associated with a chronic inflammatory response, but knowledge on the contribution of distinct IVD cells, namely CD44, to the progression of IVD degeneration remains elusive. Material: Bovine nucleus pulposus (NP) cells. Treatment: Stimulation with IL-1beta (10ng/ml). Methods: CD44 NP cells were sorted and compared by gene expression and proteomics with the negative counterpart. Dynamics of CD44 gene and protein expression was analyzed upon pro-inflammatory treatment. Results: CD44 has a multidimensional functional role in IVD metabolism, ECM synthesis and production of neuropermissive factors. CD44 widespread expression in NP has been partially associated with CD14 and CD45, resulting in the identification of distinct cell subsets. Conclusions: This study points out CD44 and CD44-based cell subsets as relevant targets in the modulation of the IVD pro-inflammatory/degenerative cascade.